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Huperzine A activates Wnt/β-catenin signaling and enhances the nonamyloidogenic pathway in an Alzheimer transgenic mouse model.
Huperzine A (HupA) is a reversible and selective inhibitor of acetylcholinesterase (AChE), and it has multiple targets when used for Alzheimer's disease (AD) therapy. In this study, we searched for new mechanisms by which HupA could activate Wnt signaling and reduce amyloidosis in AD brain. A nasal gel containing HupA was prepared. No obvious toxicity of intranasal administration of HupA was found in mice. HupA was administered intranasally to β-amyloid (Aβ) precursor protein and presenilin-1 double-transgenic mice for 4 months. We observed an increase in ADAM10 and a decrease in BACE1 and APP695 protein levels and, subsequently, a reduction in Aβ levels and Aβ burden were present in HupA-treated mouse brain, suggesting that HupA enhances the nonamyloidogenic APP cleavage pathway. Importantly, our results further showed that HupA inhibited GSK3α/β activity, and enhanced the β-catenin level in the transgenic mouse brain and in SH-SY5Y cells overexpressing Swedish mutation APP, suggesting that the neuroprotective effect of HupA is not related simply to its AChE inhibition and antioxidation, but also involves other mechanisms, including targeting of the Wnt/β-catenin signaling pathway in AD brain.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Hyaluronan and the interaction between CD44 and epidermal growth factor receptor in oncogenic signaling and chemotherapy resistance in head and neck cancer.
To investigate whether hyaluronan (HA) and CD44 (hereinafter HA-CD44) promotes head and neck squamous cell carcinoma (HNSCC) chemotherapy resistance and whether HA-CD44 promotes epidermal growth factor receptor (EGFR)-mediated oncogenic signaling to alter chemotherapy sensitivity in HNSCC. Hyaluronan, a glycosaminoglycan component of the extracellular matrix, is a ligand for the transmembrane receptor CD44, which acts through multiple signaling pathways to influence cellular behavior. We recently determined that HA-CD44 promotes phospholipase C-mediated calcium signaling and cisplatin resistance in HNSCC.Cell line study.Tumor cell growth with various chemotherapeutic drugs (methotrexate, doxorubicin hydrochloride, adriamycin, and cisplatin) was measured in the presence or absence of HA and other inhibitors of the EGFR-mediated signaling pathway. Immunoblotting was used to study EGFR signaling. Migration assays provided one measure of tumor progression.The addition of HA, but not HA plus anti-CD44 antibody, resulted in a 2-fold reduced ability of methotrexate and an 8-fold reduced ability of adriamycin to cause HNSCC cell death. Immunoblotting studies demonstrated that HA can promote an association between CD44 and EGFR as well as CD44-dependent activation of EGFR-mediated signaling. Migration assays demonstrated that HA-CD44 can promote tumor migration with EGFR signaling. The presence of AG1478, an EGFR inhibitor, and U0126, an extracellular signal-regulated kinase inhibitor, inhibited HA-mediated tumor growth, migration, and chemotherapy resistance.Our results indicate that HA promotes CD44/EGFR interaction, EGFR-mediated oncogenic signaling, and chemotherapy resistance in HNSCC. Perturbation of HA-CD44-mediated signaling may be a promising and novel strategy to treat HNSCC.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Hydroxamic acid derivatives of mycophenolic acid inhibit histone deacetylase at the cellular level.
Mycophenolic acid (MPA, 1), an inhibitor of IMP-dehydrogenase (IMPDH) and a latent PPARgamma agonist, is used as an effective immunosuppressant for clinical transplantation and recently entered clinical trials in advanced multiple myeloma patients. On the other hand, suberoylanilide hydroxamic acid (SAHA), a non-specific histone deacetylase (HDAC) inhibitor, has been approved for treating cutaneous T-cell lymphoma. MPA seemed to bear a cap, a linker, and a weak metal-binding site as a latent inhibitor of HDAC. Therefore, the hydroxamic acid derivatives of mycophenolic acid having an effective metal-binding site, mycophenolic hydroxamic acid (MPHA, 2), 7-O-acetyl mycophenolic acid (7-O-Ac MPHA, 3), and 7-O-lauroyl mycophenolic hydroxamic acid (7-O-L MPHA, 4) were designed and synthesized. All these compounds inhibited histone deacetylase with IC50 values of 1, 0.9 and 0.5 microM, and cell proliferation at concentrations of 2, 1.5 and 1 microM, respectively.
Document Type:

Reference

Product Catalog Number:

06-559
Hyperactivated FRS2α-mediated signaling in prostate cancer cells promotes tumor angiogenesis and predicts poor clinical outcome of patients.
Metastasis of tumors requires angiogenesis, which is comprised of multiple biological processes that are regulated by angiogenic factors. The fibroblast growth factor (FGF) is a potent angiogenic factor and aberrant FGF signaling is a common property of tumors. Yet, how the aberration in cancer cells contributes to angiogenesis in the tumor is not well understood. Most studies of its angiogenic signaling mechanisms have been in endothelial cells. FGF receptor substrate 2α (FRS2α) is an FGF receptor-associated protein required for activation of downstream signaling molecules that include those in the mitogen-activated protein and AKT kinase pathways. Herein, we demonstrated that overactivation and hyperactivity of FRS2α, as well as overexpression of cJUN and HIF1α, were positively correlated with vessel density and progression of human prostate cancer (PCa) toward malignancy. We also demonstrate that FGF upregulated the production of vascular endothelial growth factor A mainly by increasing expression of cJUN and HIF1α. This then promoted recruitment of endothelial cells and vessel formation for the tumor. Tumor angiogenesis in mouse PCa tissues was compromised by tissue-specific ablation of Frs2α in prostate epithelial cells. Depletion of Frs2α expression in human PCa cells and in a preclinical xenograft model, MDA PCa 118b, also significantly suppressed tumor angiogenesis accompanied with decreased tumor growth in the bone. The results underscore the angiogenic role of FRS2α-mediated signaling in tumor epithelial cells in angiogenesis. They provide a rationale for treating PCa with inhibitors of FGF signaling. They also demonstrate the potential of overexpressed FRS2α as a biomarker for PCa diagnosis, prognosis and response to therapies.
Document Type:

Reference

Product Catalog Number:

17-371
Product Catalog Name:

EZ-ChIP™
Hypermethylated in cancer 1 (HIC1), a tumor suppressor gene epigenetically deregulated in hyperparathyroid tumors by histone H3 lysine modification.
Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance.The aim of the study was to investigate whether HIC1 may act as a tumor suppressor in the parathyroid glands and whether deregulated expression involves epigenetic mechanisms.Parathyroid tumors from patients with pHPT included single adenomas, multiple tumors from the same patient, and cancer. Hyperplastic parathyroid glands from patients with sHPT and hypercalcemia and normal parathyroid tissue specimens were included in the study. Quantitative RT-PCR, bisulfite pyrosequencing, colony formation assay, chromatin immunoprecipitation, and RNA interference was used.HIC1 was generally underexpressed regardless of the hyperparathyroid disease state including multiple parathyroid tumors from the same patient, and overexpression of HIC1 led to a decrease in clonogenic survival of parathyroid tumor cells. Only the carcinomas showed a high methylation level and reduced HIC1 expression. Cell culture experiments, including use of primary parathyroid tumor cells prepared directly after operation, the general histone methyltransferase inhibitor 3-deazaneplanocin A, chromatin immunoprecipitation, and RNA interference of DNA methyltransferases and EZH2 (enhancer of zeste homolog 2), supported a role of repressive histone H3 modifications (H3K27me2/3) rather than DNA methylation in repression of HIC1.The results strongly support a growth-regulatory role of HIC1 in the parathyroid glands and suggest that perturbed expression of HIC1 may represent an early event during tumor development. Repressive histone modification H3K27me2/3 is involved in repression of HIC1 expression in hyperparathyroid tumors.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Hypoxia upregulates Rab11-family interacting protein 4 through HIF-1α to promote the metastasis of hepatocellular carcinoma.
Hypoxic microenvironment is a powerful driving force for the invasion and metastasis of hepatocellular carcinoma (HCC). Hypoxia-inducible factor 1α (HIF-1α), as a crucial regulator of transcriptional responses to hypoxia, induces the expression of multiple target genes involved in different steps of HCC metastatic process. It is critical to find target genes associated with metastasis under hypoxia for shedding new light on molecular mechanism of HCC metastasis. In this study, we uncovered that hypoxia could induce the upregulation of Rab11-family interacting protein 4 (Rab11-FIP4) and activation of Rab11-FIP4 promoter by HIF-1α. The overexpression of Rab11-FIP4 significantly enhanced the mobility and invasiveness of HCC cells in vitro, also contributed to distant lung metastasis in vivo, whereas silencing of Rab11-FIP4 decreased the ability of migration and invasion in HCC cells in vitro and suppressed lung metastasis in vivo. Rab11-FIP4 facilitated HCC metastasis through the phosphorylation of PRAS40, which was regulated by mTOR. Furthermore, the expression level of Rab11-FIP4 was significantly increased in HCC tissues and high expression of Rab11-FIP4 was closely correlated with vascular invasion and poor prognosis in HCC patients. A markedly positive correlation between the expression of Rab11-FIP4 and HIF-1α was observed in HCC tissues and combination of Rab11-FIP4 and HIF-1α was a more valuable predictor of poor prognosis for HCC patients. In conclusion, Rab11-FIP4 is a target gene of HIF-1α and has a pro-metastatic role in HCC, suggesting that Rab11-FIP4 may be a promising candidate target for HCC treatment.
Document Type:

Reference

Product Catalog Number:

17-371
Product Catalog Name:

EZ-ChIP™
IBM1, a JmjC domain-containing histone demethylase, is involved in the regulation of RNA-directed DNA methylation through the epigenetic control of RDR2 and DCL3 expressi ...
Small RNA-directed DNA methylation (RdDM) is an important epigenetic pathway in Arabidopsis that controls the expression of multiple genes and several developmental processes. RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3) are necessary factors in 24-nt small interfering RNA (siRNA) biogenesis, which is part of the RdDM pathway. Here, we found that Increase in BONSAI Methylation 1 (IBM1), a conserved JmjC family histone demethylase, is directly associated with RDR2 and DCL3 chromatin. The mutation of IBM1 induced the hypermethylation of H3K9 and DNA non-CG sites within RDR2 and DCL3, which repressed their expression. A genome-wide analysis suggested that the reduction in RDR2 and DCL3 expression affected siRNA biogenesis in a locus-specific manner and disrupted RdDM-directed gene repression. Together, our results suggest that IBM1 regulates gene expression through two distinct pathways: direct association to protect genes from silencing by preventing the coupling of histone and DNA methylation, and indirect silencing of gene expression through RdDM-directed repression.
Document Type:

Reference

Product Catalog Number:

04-745
Product Catalog Name:

Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal
Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation.
Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.
Document Type:

Reference

Product Catalog Number:

07-760
Product Catalog Name:
Identification and expression analysis of connexin-45 and connexin-60 as major connexins in porcine oocytes.
During mammalian oogenesis, intercellular communication between oocytes and the surrounding follicle cells through gap junction channels is crucial for oocyte development and maturation. The channel properties of gap junctions may be affected by the composition or combination of connexins, the expression of which is regulated by gonadotropins and other factors. Thus, identification and expression analysis of connexin genes in oocytes and follicle cells will help us to better understand how oogenesis and folliculogenesis are regulated in a species-specific manner in mammals. We previously reported the spatiotemporal expression of multiple connexin genes in porcine follicle cells. Here, we searched for connexin genes specifically expressed in porcine oocytes that may be involved in the formation of gap junctions between oocytes and follicle cells. To achieve this, we constructed an oocyte-specific cDNA library to identify which connexin genes are expressed in these cells and found that gap junction protein, alpha 10, which encodes connexin-60, and a porcine ortholog of mouse gap junction protein, gamma 1 encoding connexin-45, are the major connexins expressed in porcine oocytes during folliculogenesis. Immunostaining and in situ hybridization of sectioned porcine ovaries confirmed oocyte expression of these genes at 3 different stages of ovary development. Furthermore, their gap junction channel activity was assessed using a heterologous cell system. However, gap junction protein, alpha 4, which encodes connexin-37 and is expressed in the oocytes of several other mammals, was undetectable. We demonstrate that there is diversity in the connexin genes expressed in mammalian oocytes, and hence in the gap junctions connecting oocytes and cumulus cells.
Document Type:

Reference

Product Catalog Number:

AB1745
Product Catalog Name:

Anti-Connexin 45 Antibody, near CT, cytoplasmic
Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.
Deletions involving regions of chromosome 10 occur in the vast majority (> 90%) of human glioblastoma multiformes. A region at chromosome 10q23-24 was implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. We have identified a gene, designated MMAC1, that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. Our results identify a strong candidate tumour suppressor gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers.
Document Type:

Reference

Product Catalog Number:

MAB4037
Product Catalog Name:

Anti-PTEN Antibody, CT, clone A2b1
Identification of a chronic obstructive pulmonary disease genetic determinant that regulates HHIP.
Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Identification of a face enhancer reveals direct regulation of LIM homeobox 8 (Lhx8) by wingless-int (WNT)/β-catenin signaling.
Development of the mammalian face requires a large number of genes that are expressed with spatio-temporal specificity, and transcriptional regulation mediated by enhancers plays a key role in the precise control of gene expression. Using chromatin immunoprecipitation for a histone marker of active enhancers, we generated a genome-wide map of candidate enhancers from the maxillary arch (primordium for the upper jaw) of mouse embryos. Furthermore, we confirmed multiple novel craniofacial enhancers near the genes implicated in human palate defects through functional assays. We characterized in detail one of the enhancers (Lhx8_enh1) located upstream of Lhx8, a key regulatory gene for craniofacial development. Lhx8_enh1 contained an evolutionarily conserved binding site for lymphoid enhancer factor/T-cell factor family proteins, which mediate the transcriptional regulation by the WNT/β-catenin signaling pathway. We demonstrated in vitro that WNT/β-catenin signaling was indeed essential for the expression of Lhx8 in the maxillary arch cells and that Lhx8_enh1 was a direct target of the WNT/β-catenin pathway. Together, we uncovered a molecular mechanism for the regulation of Lhx8, and we provided valuable resources for further investigation into the gene regulatory network of craniofacial development.
Document Type:

Reference

Product Catalog Number:

12-370
Product Catalog Name:

Normal Rabbit IgG
Identification of a QTL in Mus musculus for alcohol preference, withdrawal, and Ap3m2 expression using integrative functional genomics and precision genetics.
Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with greater than 60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits.
Document Type:

Reference

Product Catalog Number:

07-473
Product Catalog Name:

Anti-trimethyl-Histone H3 (Lys4) Antibody
Identification of a role for histone H2B ubiquitylation in noncoding RNA 3'-end formation through mutational analysis of Rtf1 in Saccharomyces cerevisiae.
The conserved eukaryotic Paf1 complex regulates RNA synthesis by RNA polymerase II at multiple levels, including transcript elongation, transcript termination, and chromatin modifications. To better understand the contributions of the Paf1 complex to transcriptional regulation, we generated mutations that alter conserved residues within the Rtf1 subunit of the Saccharomyces cerevisiae Paf1 complex. Importantly, single amino acid substitutions within a region of Rtf1 that is conserved from yeast to humans, which we termed the histone modification domain, resulted in the loss of histone H2B ubiquitylation and impaired histone H3 methylation. Phenotypic analysis of these mutations revealed additional defects in telomeric silencing, transcription elongation, and prevention of cryptic initiation. We also demonstrated that amino acid substitutions within the Rtf1 histone modification domain disrupt 3'-end formation of snoRNA transcripts and identify a previously uncharacterized regulatory role for the histone H2B K123 ubiquitylation mark in this process. Cumulatively, our results reveal functionally important residues in Rtf1, better define the roles of Rtf1 in transcription and histone modification, and provide strong genetic support for the participation of histone modification marks in the termination of noncoding RNAs.
Document Type:

Reference

Product Catalog Number:

07-030
Product Catalog Name:

Anti-dimethyl-Histone H3 (Lys4) Antibody
Identification of an histone H3 acetylated/K4-methylated-bound intragenic enhancer regulatory for urokinase receptor expression.
The transcriptionally regulated urokinase-type plasminogen activator receptor (u-PAR) contributes to cancer progression. Although previous studies have identified multiple 5' regulatory elements, these cis motifs cannot fully account for u-PAR expression prompting a search for hitherto uncharacterized regulatory elements. DNase I hypersensitivity and chromatin immunoprecipitation assays using u-PAR-expressing colon cancer cells indicated a hypersensitive region (+665/+2068) in intron 1 enriched with acetylated histone 3 (H3) and H3 methylated at lysine 4, markers of regulatory regions. The +665/+2068 region increased transcription from a u-PAR-promoter in an orientation- and distance-independent manner fulfilling the criteria of an enhancer. Optimal stimulation of the u-PAR promoter by phorbol ester required this enhancer. Systematic truncations combined with DNase I footprinting revealed two protected regions (+1060/+1099 and +1123/+1134) with deletion of the latter practically abolishing enhancer activity. The +1123/+1134 region harbored non-consensus activator protein-1 and Ets1 binding sites bound with c-Jun (and/or the related JunD/JunB) and c-Fos (and/or the related FosB/Fra-1/Fra-2) as revealed with chromatin immunoprecipitation. Further, nuclear extract from resected colon cancers showed elevated protein binding to a +1123/+1134-spanning probe coordinate with elevated u-PAR protein. Thus, we have defined a novel intragenic enhancer in the u-PAR gene required for constitutive and inducible expression.
Document Type:

Reference

Product Catalog Number:

06-599
Product Catalog Name:

Anti-acetyl-Histone H3 Antibody
Identification of an intrinsic determinant critical for maspin subcellular localization and function.
Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1). Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL) and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate(346) (D(346)) to Glutamate (E(346)), maspin(D346E), was predominantly nuclear, whereas wild type maspin (maspin(WT)) was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC) showed that the increased nuclear enrichment of maspin(D346E) was, at least in part, due to its increased affinity to HDAC1. Maspin(D346E) was also more potent than maspin(WT) as an HDAC inhibitor. Taken together, our evidence demonstrates that D(346) is a critical cis-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Identification of Arx transcriptional targets in the developing basal forebrain.
Mutations in the aristaless-related homeobox (ARX) gene are associated with multiple neurologic disorders in humans. Studies in mice indicate Arx plays a role in neuronal progenitor proliferation and development of the cerebral cortex, thalamus, hippocampus, striatum, and olfactory bulbs. Specific defects associated with Arx loss of function include abnormal interneuron migration and subtype differentiation. How disruptions in ARX result in human disease and how loss of Arx in mice results in these phenotypes remains poorly understood. To gain insight into the biological functions of Arx, we performed a genome-wide expression screen to identify transcriptional changes within the subpallium in the absence of Arx. We have identified 84 genes whose expression was dysregulated in the absence of Arx. This population was enriched in genes involved in cell migration, axonal guidance, neurogenesis, and regulation of transcription and includes genes implicated in autism, epilepsy, and mental retardation; all features recognized in patients with ARX mutations. Additionally, we found Arx directly repressed three of the identified transcription factors: Lmo1, Ebf3 and Shox2. To further understand how the identified genes are involved in neural development, we used gene set enrichment algorithms to compare the Arx gene regulatory network (GRN) to the Dlx1/2 GRN and interneuron transcriptome. These analyses identified a subset of genes in the Arx GRN that are shared with that of the Dlx1/2 GRN and that are enriched in the interneuron transcriptome. These data indicate Arx plays multiple roles in forebrain development, both dependent and independent of Dlx1/2, and thus provides further insights into the understanding of the mechanisms underlying the pathology of mental retardation and epilepsy phenotypes resulting from ARX mutations.
Document Type:

Reference

Product Catalog Number:

MAB374
Product Catalog Name:

Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5
Identification of chimeric TSNAX-DISC1 resulting from intergenic splicing in endometrial carcinoma through high-throughput RNA sequencing.
Gene fusion is among the primary processes that generate new genes and has been well characterized as potent pathway of oncogenesis. Here, by high-throughput RNA sequencing in nine paired human endometrial carcinoma (EC) and matched non-cancerous tissues, we obtained that chimeric translin-associated factor X-disrupted-in-schizophrenia 1 (TSNAX-DISC1) occurred significantly upregulated in multiple EC samples. Experimental investigation showed that TSNAX-DISC1 appears to be formed by splicing without chromosomal rearrangement. The chimera expression inversely correlated with the binding of CCCTC-binding factor (CTCF) to the insulators. Subsequent investigations indicate that long intergenic non-coding RNA lincRNA-NR_034037, separating TSNAX from DISC1, regulates TSNAX -DISC1 production and TSNAX/DISC1 expression levels by extricating CTCF from insulators. Dysregulation of TSNAX influences steroidogenic factor-1-stimulated transcription on the StAR promoter, altering progesterone actions, implying the association with cancer. Together, these results advance our understanding of the mechanism in which lincRNA-NR_034037 regulates TSNAX-DISC1 formation programs that tightly regulate EC development.
Document Type:

Reference

Product Catalog Number:

17-700
Product Catalog Name:

Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit
Identification of direct regulatory targets of the transcription factor Sox10 based on function and conservation.
Sox10, a member of the Sry-related HMG-Box gene family, is a critical transcription factor for several important cell lineages, most notably the neural crest stem cells and the derivative peripheral glial cells and melanocytes. Thus far, only a handful of direct target genes are known for this transcription factor limiting our understanding of the biological network it governs.We describe identification of multiple direct regulatory target genes of Sox10 through a procedure based on function and conservation. By combining RNA interference technique and DNA microarray technology, we have identified a set of genes that show significant down-regulation upon introduction of Sox10 specific siRNA into Schwannoma cells. Subsequent comparative genomics analyses led to potential binding sites for Sox10 protein conserved across several mammalian species within the genomic region proximal to these genes. Multiple sites belonging to 4 different genes (proteolipid protein, Sox10, extracellular superoxide dismutase, and pleiotrophin) were shown to directly interact with Sox10 by chromatin immunoprecipitation assay. We further confirmed the direct regulation through the identified cis-element for one of the genes, extracellular superoxide dismutase, using electrophoretic mobility shift assay and reporter assay.In sum, the process of combining differential expression profiling and comparative genomics successfully led to further defining the role of Sox10, a critical transcription factor for the development of peripheral glia. Our strategy utilizing relatively accessible techniques and tools should be applicable to studying the function of other transcription factors.
Document Type:

Reference

Product Catalog Number:

17-371
Product Catalog Name:

EZ-ChIP™
Identification of downstream-initiated c-Myc proteins which are dominant-negative inhibitors of transactivation by full-length c-Myc proteins.
The c-myc gene has been implicated in multiple cellular processes including proliferation, differentiation, and apoptosis. In addition to the full-length c-Myc 1 and 2 proteins, we have found that human, murine, and avian cells express smaller c-Myc proteins arising from translational initiation at conserved downstream AUG codons. These c-Myc short (c-Myc S) proteins lack most of the N-terminal transactivation domain but retain the C-terminal protein dimerization and DNA binding domains. As with full-length c-Myc proteins, the c-Myc S proteins appear to be localized to the nucleus, are relatively unstable, and are phosphorylated. Significant levels of c-Myc S, often approaching the levels of full-length c-Myc, are transiently observed during the rapid growth phase of several different types of cells. Optimization of the upstream initiation codons resulted in greatly reduced synthesis of the c-Myc S proteins, suggesting that a "leaky scanning" mechanism leads to the translation of these proteins. In some hematopoietic tumor cell lines having altered c-myc genes, the c-Myc S proteins are constitutively expressed at levels equivalent to that of full-length c-Myc. As predicted, the c-Myc S proteins are unable to activate transcription and inhibited transactivation by full-length c-Myc proteins, suggesting a dominant-negative inhibitory function. While these transcriptional inhibitors would not be expected to function as full-length c-Myc, the occurrence of tumors which express constitutive high levels of c-Myc S and their transient synthesis during rapid cell growth suggest that these proteins do not interfere with the growth-promoting functions of full-length c-Myc.
Document Type:

Reference

Product Catalog Number:

06-340
Product Catalog Name:

Anti-Myc Antibody